1. Field of the Invention
The present invention relates generally to novel quinobenzoxazines, methods of synthesis, and uses thereof. More particularly, it concerns the synthesis of quinobenzoxazine compounds, pyridobenzophenoxazines, pyrridonaphthophenoxazines, and other related compounds that can stabilize the topoisomerase II-DNA complex and interact with G-quadruplex DNA and that, as a consequence, exhibit anticancer and antibiotic activity.
2. Description of Related Art
There remains a persistent need for new compounds with antibiotic properties. Over time bacterial agents evolve to develop resistance to existing classes of antibiotics, thus driving the continual need for new and effective antibiotics. The development of new classes of compounds with both narrow and broad spectrum antibacterial properties is therefore desired.
Similarly, there also has been a need for, and much research focused on, the treatment of cancer using chemotherapies. Resources continue to be directed toward the development of antineoplastic agents for the treatment of cancers, including solid tumors, leukemias, and other forms of cancer. Many antineoplastic agents developed are not ideal because of problems associated with their cytotoxicity and multidrug resistance of some cancers. What is needed are compositions for the treatment of cancers that are effective while minimizing their adverse effects.
The cytotoxicity of the fluoroquinolones is due to their ability to shift the cleavage-religation equilibrium required for topoisomerase action toward cleavage, thereby effectively trapping the enzyme on DNA to form the xe2x80x9ccleaved complexxe2x80x9d (Shen et al., 1989a; Shen et al., 1989b; Shen et al., 1989c; Willmott and Maxwell, 1993). The quinobenzoxazines are potent mammalian DNA topoisomerase II inhibitors. It has been proposed that the quinobenzoxazines inhibit DNA topoisomerase II reaction at a step prior to the formation of the xe2x80x9ccleaved complexxe2x80x9d intermediate (Permana et al., 1994).
The quinobenzoxazines are synthetic analogues of antibacterial fluoroquinolones (Chu and Maleczka, Jr., 1987; Chu et al., 1992; Chu et al., 1994). Studies have shown that some quinobenzoxazine derivatives have curative activity against solid tumors, murine tumors, and human xenographs (Clement et al., 1995). The antineoplastic effects of related compounds, the quinobenzothiazines, are detailed in U.S. Pat. No. 5,624,924, and is incorporated herin by reference. DNA binding studies reveal that antibacterial fluoroquinolones prefer to bind single-stranded DNA to duplex DNA or bind the DNA-gyrase complex in the presence of Mg2+ (Willmott and Maxwell, 1993). In contrast, the quinobenzoxazines bind duplex DNA through intercalation. A drug self-assembly model has been proposed for the quinobenzoxazines based on results of biophysical and biochemical studies (Fan et al., 1995; Yu et al., 1996). In this model, a 2:2 drug:Mg2+ dimer binds DNA with one drug molecule intercalating between DNA base pairs and the other drug molecule externally bound through two chelated Mg2+ ions. The two magnesium cations link the two drug molecules in a head-to-tail fashion in which the xcex2-ketoacid moiety is the bidentate ligand as a head and the primary amino group of the amino-pyrrolidine side chain is the tail. Each magnesium cation also binds with one phosphate oxygen of DNA backbone and two water molecules to form an octahedral complex.
The model interacts with DNA as follows: the polyaromatic ring of one moiety intercalates into DNA base pairs and anchors the whole assembly on DNA; and the xcex2-ketoacid functionality and the 3-amino-pyrrolidine substituent of the second moiety chelate Mg2+ through which the external molecule is bound in the DNA minor groove. In this manner, one quinobenzoxazine molecule serves as a DNA intercalator and the other quinobenzoxazine molecule binds externally, held to the first drug molecule by two Mg2+ ions (Fan et al., 1995). On the basis of this model, new combinations of antibacterials and anticancer agents can be designed that target the bacterial gyrase-DNA and topoisomerase II-DNA complexes, respectively. In each case, one molecule designed to optimally interact with the DNA by intercalation, and the other designed to interact with the enzyme by external interaction with the DNA, are predicted to produce greater efficacy than the same molecule serving both roles in a suboptimum manner. G-quadruplexes also have been proposed as molecular targets for anticancer agents (Mergny and Hxc3xa8lxc3xa9ne, 1998). Drug interaction with G-quadruplexes leads to inhibition of telomerase (Sun et al., 1997; Wheelhouse et al., 1998; Fedoroff et al., 1998). Inhibition of telomerase has been proposed as a selective way to kill cancer cells because in large part only cancer cells depend upon telomerase for survival (Morin, 1995; Parkinson, 1996; Raymond et al., 1996).
Single agents that have dual mechanisms of action are proposed to have an advantage over agents that only have one defined mechanism of action. For example, studies have shown that Taxol not only targets tubulin, but also interacts with bcl-2 (Rodi, 1999). This may account for some of the efficacy of Taxol in its anticancer activity.
The present invention provides a new and novel solid- and solution-state parallel synthesis method for quinobenzoxazine analogues, a novel model of quinobenzoxazine self-assembling on DNA, and use of this model to design a series of new quinobenzoxazines, pyridobenzophenoxazines, pyrridonaphthophenoxazines and other related compounds that exhibit anticancer or antibiotic activity. The anticancer activity of these novel compounds is thought to operate via stabilization of the topoisomerase II-DNA complex and/or interaction with G-quadruplexes. The antibiotic activity of these compounds derives from their ability to interact with the gyrase-DNA complex, which is the bacterial type II topoisomerase.
The present invention therefore seeks to overcome deficiencies in the prior art by providing a new model for design and a new method for the synthesis, of a new series of quinobenzoxazines that display increased anticancer and antibiotic activities. One aspect of the invention includes a novel dimer model compound for use in developing quinobenzoxazines with potentiated anticancer and antibiotic activity. Further provided is a solid-state parallel synthesis method of producing these antineoplastic quinobenzoxazines and quinobenzoxazine derivatives.
The present invention contemplates a 2:2 quinobenzoxazine:Mg2+ dimer model. This dimer binds DNA with one drug molecule intercalating between DNA base pairs and the other drug molecule externally binds through two chelated Mg2+ ions. The magnesium cations link two drug molecules in a head-to-tail fashion in which the xcex2-ketoacid moiety is the bidentate ligand as a head and the primary amino group of the amino pyrrolidine side chain is the tail. Each magnesium cation also binds with a single phosphate oxygen of the DNA backbone and two water molecules to form an octahedral complex. In this manner, one quinobenzoxazine molecule of the dimer serves as a DNA intercalator and the other quinobenzoxazine molecule binds externally, held to the first drug molecule by two Mg2+ ions.
Alternatively, the 2:2 quinobenzoxazine:Mg2+ dimer model utilizes a heterodimer. Based on the fact that fluoroquinolones, parent compounds to quinobenzoxazines, in the presence of quinobenzoxazines, can cooperatively interact with DNA in the presence of magnesium and have a cooperative effect on the lengthening of the DNA helix, a heterodimer model was made. The heterodimer is constructed by removal of the externally bound quinobenzoxazine molecule and substituting the nonintercalating fluoroquinolone parent molecule. The intercalating aspect of the dimer model remains a quinobenzoxazine. Upon addition of the nonintercalating fluoroquinolone to the quinobenzoxazine, a cooperative effect on DNA lengthening is observed that is consistent with the substitution of the externally bound quinobenzoxazine by the nonintercalating fluoroquinolone. This result provides useful information in that it reveals that both the intercalating and the externally bound moieties, as well as the Mg2+ binding ability, of the quinobenzoxazine are useful in the design and synthesis of novel quinobenzoxazines with potentiated anticancer and antibiotic activity. Furthermore, the inventors demonstrate that this heterodimer model also exists in the presence of topoisomerase II bound to DNA. In an in vitro study using a prostate cancer cell line, it was found that addition of the nonintercalating fluoroquinolone Norfloxacin increased the potency of the quinobenzoxazine up to 20-fold, providing additional evidence for the 2:2 quinobenzoxazine:Mg2+ dimer model.
In addition to topoisomerase II as a molecular target, some of the quinobenzoxazines interact with G-quadruplexes. As a result of G-quadruplex interaction, these compounds inhibit telomerase and cause chromosomal aberrations. Thus, in this present invention there are three categories of anticancer drugs. The first two groups of compounds are those that are either topoisomerase II interactive or G-quadruplex interactive, while the third group can interact with both targets.
The present invention further provides a novel method of solid-based parallel synthesis of quinobenzoxazines. The first step is the loading of a quinobenzoxazine precursor onto a solid support to provide a solid bound ester. A variety of solid supports may be utilized. Virtually any solid support that is capable of binding to a precursor to form a solid bound ester through a transesterification reaction is acceptable so long as the solid support is compatible with the protective groups used during peptide synthesis. These protective groups include Fmoc, t-Boc, and Teoc. The protective groups may be readily removed using conditions suitable for the particular group employed. For example, Fmoc is removed under basic conditions, t-Boc is removed under acidic conditions, and Teoc is removed in the presence of fluoride ion. Thus, for an acceptable solid support to work with the instant invention, it must contain properties that allow the growing peptide chains to be protected or deprotected as desired under appropriate conditions. In a preferred embodiment, a Wang resin is used as a solid support to which the precursor is bound by a transesterification reaction in the presence of a catalyst. Generally, any carboxyacetophenone containing fluorine atoms or nitro groups on the two and three positions of the benzene ring may be employed. These compounds may be modified using combinatorial chemistry techniques to produce a tetracyclic quinobenzoxazine scaffolding, which also can undergo a transesterification reaction to bind to the resin or other solid support. A preferred precursor is ethyl 2,3,4,5-tetrafluorobenzoacetate.
The next step in the disclosed solid-state synthesis comprises a generation of enaminoketoester from the solid bound ester formed during the transesterification reaction outlined above. This is considered a branch point from which diversified scaffolding can be made.
The invention thus provides for the formation of a tetracyclic scaffolding via a double cyclization step from the enaminoketoester. In a preferred embodiment, the organic base is tetramethylguanidine (TMG).
After formation of the scaffolding, the fluorine at the 7 position may be substituted by a nucleophilic (for example, a nitrogeneous base), thereby generating any of a wide variety of quinobenzoxazine analogues.
Once the quinobenzoxazine analogues have been formed by regio-specific substitution of the fluorine further derivatization may be accomplished by deprotection of the benzylic amino groups and subsequent coupling with an amino acid, acylating agent, or alkylating agent. The coupling agents are selected on the basis of their potential to exhibit increased DNA unwinding and DNA binding properties as compared with the parent quinobenzoxazines. One may select for these properties by first testing candidates in the dimer model previously discussed. It is demonstrated that benzo-annulated quinobenzoxazines will exhibit enhanced DNA binding and/or increased DNA unwinding compared with quinobenzoxazines.
Once a desired compound is synthesized by the disclosed method, the final step of the solid-state synthesis method is to cleave the compounds from the solid support.
The invention further provides novel quinobenzoxazines and quinobenzoxazine analogues. These molecules may be designed by use of the dimer model described and synthesized by the solid-state synthesis method disclosed herein.
It is understood that any benzo-annulated quinobenzoxazine, or any quinobenzoxazine containing a quinobenzoxazine with increased DNA binding or unwinding capacities, is encompassed by the instant invention. Particularly useful compounds are those with benzo-annulated ring systems and extended side chains.